1. Field of the Invention
The present disclosure relates to a method for preparing recombinant glycoproteins with high sialic acid content.
2. Description of the Related Art
Sialic acid (Sia, NeuAc, NeuGc) is a generic term for acyl derivatives of neuraminic acid (Neu). In 1936, sialic acid was first isolated by Blix from mucine of bovine salivary gland and is an acidic sugar which is composed of 9 carbons and has a COOH group. According to differences in substitution group, 50 types of sialic acids have been reported (Angata, T. and Varki, A. Chem. Rev 102, 439-469, 2002), which are known to be specifically distributed in species and tissues. In higher animals, sialic acid is linked to Gal, GlcNAc, GalNAc and sialic acid of glycoprotein, glycolipid, and oligosaccharide glycans by reaction of a specific sialyltransferase, respectively, through alpha-glycosidic bonding.
Since sialic acid of glycoconjugate glycan is located at the very end of a glycan structure present on a surface of a cell membrane, it has been expected to be directly involved in contact between the cell and extracellular environment, and it has been known for awhile that the lifespan of blood cells or glycoprotein in body fluid is shortened by removal of sialic acid. For example, when sialic acid on red blood cell membrane is removed (asialylation), galactose is exposed on a cell surface and binds to a receptor lectin, which specifically binds to galactose on a Kupffer cell surface. Thereby, galactose is removed from a circulatory system by a receptor-mediated endocytosis, and asialoglycoprotein, from which sialic acid is removed, is also bound by lectin on a hepatocyte surface and is removed from a circulatory system in a similar pathway to the red blood cell. Furthermore, for alpha-antitrypsin, cholinesterase, chorionic gonadotropin, CTLA4Ig, Factor VIII, gamma-glutamyltransferase, granulocyte colony-stimulating Factor (G-CSF) and luteinizing hormone (LH), which are sialylated glycoproteins, sialic acid-bound glycoprotein is reported to experience a significant increase in half life thereof, compared to sialic acid unbound glycoprotein (Ngantung F A. et al., 2006, Biotechnol. Bioeng 95(1), 106-119).
In particular, among sialylated glycoproteins, erythropoietin is a glycoprotein hormone which induces red blood cell generation, and a recombinant erythropoietin is being used as a therapeutic agent for anemia. Wild-type erythropoietin includes three N-glycans and one O-glycan. Since a maximum of four sialic acids may bind to one N-glycan and two sialic acids may bind to one O-glycan, potentially, 14 sialic acids in total may bind to one molecule of erythropoietin. Glycan-bound sialic acid blocks binding of asialoglycoprotein receptor present in the liver so as to prevent decomposition of erythropoietin in the liver.
Thrombopoietin (TPO) is a hormone similar to EPO produced mainly by the liver and the kidney that regulates the production of platelets in the bone marrow. TPO consists of 332 amino acids, has molecular weight approximately of 80˜100 kDa, and has six N-glycans and 24 O-glycans. The first 155 amino acids are very similar to EPO, and as in the case of the glycoprotein like EPO, the sialic acid content of glycan significantly influences in vivo stability of the protein. The above was confirmed by the observance of noticeable drop of in vivo activity of TPO from which all sialic acid was removed by sialidase (Takeuchi M, et al., 1989, Poc Natl Acad Sci, 7819-22). Like the EPO, the structure of the glycan has the tetra-antennary, tetra-sialylation and core-fucosylation forms, and in the case of recombinant human TPO, various glycan structures have the di-antennary or heterogenous forms (Inous N, et al., 1999, Glycoconjugate Journal, 16, 707-718).
Accordingly, the greater the increase in sialic acid content of glycoprotein, the greater the half life of glycoprotein in a body becomes. (Fukuda, M. N. et al., 1989, Blood 73, 84-89; Sinclair, A. M. et al, 2005, J. Pharm. Sci. 94, 1626-1635) Therefore, an increase in sialic acid content is needed for quality and bioequivalence of therapeutic glycoprotein.
Accordingly, the present inventors investigated a method for increasing internal activity by increasing sialic acid content of sialylated glycoprotein such as erythropoietin and thrombopoietin. As a result, the inventors completed the present invention by identifying a significant increase in sialic acid content of erythropoietin and thrombopoietin compared to a wild type when point mutationinduced UDP-GlcNAc 2-epimerase/ManNAc kinase(GNE/MNK), CMP-sialic acid transporter (CMP-SAT) and alpha-2,3-sialyltransferase are simultaneously overexpressed in a cell producing human erythropoietin or thrombopoietin.